VALUE = global median normalized log2 ratio (Cy5/Cy3) representing test/reference
PREHYBRIDIZATION: 1. Make 100 ml prehybridization buffer (for a maximum of 4 slides) containing 1M Tris-HCL pH=9 pre-heated at 50ºC, 100mM ethanolamide and 0.1% SDS. 2. Heat the prehybridization solution to 50 ºC in a 100 ml Coplin Jar. 3. Place slides to be analyzed into the pre-heated pre-hybridization buffer. Incubate for 20 minutes at 50 ºC with constant agitation, if possible. 4. Wash thoroughly by inverting or gently shaking the container with nuclease-free water constantly for 1 minute. Decant the water and repeat this rising step with fresh nuclease-free water. 5. Allow the slides to dry by centrifuging 3 minute 800 rpm. Slides should be used immediately following prehybridization. HYBRIDIZATION: Pre-heat 3x hybridization solution (Ambion) to 65 ºC for at least 5 min to overcome SDS precipitation. Combine target (moDC 0h- and 16h-post LPS stimulation labelled RNA) with hybridization buffer (preheated to 65 ºC). Heat at 95 ºC for 3 minutes. Immediately put on ice for at least 1 min. Spin-down briefly. Add 4x blocking reagent (Kreatech). Incubate on dark for 1 minute at room temperature. Using Agilent gasket slides (G2534-60003), Agilent SureHyb hybridization chambers (G2534A) and hybridization oven (G2545A), see Agilent User Manual for instructions: 1. For each array, put a gasket slide onto a SureHyb chamber and add a total of 250 ul target as 1 dot in the center of the chamber. 2. Put a microarray slide on the gasket slide by holding it in place with your fingertips, towards the top edge of the gasket chamber. 3. Gently (!) allow the microarray slide to drop onto the gasket chamber by lowering it with the aid of your other hand. Do not move the microarray slide after it is in place. 4. Place the SureHyb chamber into the hybridization oven at 42 ºC for 16 hours.
Pre-processing of the data was performed using BRB-ArrayTools v3.4.0 software. Manually flagged bad spots were eliminated and the local background was subtracted, followed by the averaging of replicate features on the array. Data points were removed when intensities values for both dyes were below 200% of background. Feature extraction was performed with QuantArray v3.0 software, using the adaptative circle method for measuring the Median signal intensity and Median background intensity.For background calculations were considered the 5-20 percentile of least intense pixels and for signal calculations the 80-95 percentile of most intense pixels. The feature extraction grid was adjusted manually and visually bad spots were flagged using the software flag tool. A global median normalization of microarray data was applied and log2 intensity ratios (M values) were obtained.
Scanning of slides using the Agilent G2565AA DNA microarray scanner: Resolution: 10 um Red laser PMT= 100%, Green laser PMT= 100%
Total RNA was extracted using the miRVanaTM miRNA isolation kit (Ambion). Briefly, tissues were disrupted in lysis solution using a homogeneizer and miRNA homogenate additive added. An acid phenol:chloroform extraction was then performed followed by a filter cartridge purification.RNA quantity and quality was assessed using the Nanodrop and Agilent 2100 bioanalyzer systems, respectively. Samples with a RIN number above 7 were used in the study.
Total RNA was labelled using the miRacULS II miRNA labelling kit (Kreatech) according manufacture’s instructions. Briefly, 3 µg of total RNA from test and reference (pool of 4 First Choice Human Cervix RNAs, Ambion) samples were incubated with Cy5- and Cy3-ULS, respectively for 15 min at 85ºC. The labelled RNAs were purified to remove non-reacted Cy-ULS. Dye incorporation was monitored by UV-visible spectroscopy.